Herpes simplex virus (HSV) infection continues at epidemic levels in the global human population with serious health consequences. Intervention of the reactivation of HSV from the latent state is a critical component of efforts to control this epidemic. The long-term goal of the proposed research is to acquire knowledge of the molecular mechanisms controlling the transition from latent to lytic phase transcription for use in the rational design of intervention strategies. It is widely accepted that the viral/host cell interactions initiating the viral lytic cascade during primary infection are distinct from those initiating the lytic cascade from the latent viral genome. This notion is rooted in the fact that the alpha gene transinducing factor (VP16) is brought in with the virion as a protein and not made until late in the lytic cycle when it is incorporated as protein into the virion. Thus this important viral transactivator is thought to be absent during latency as well as during activation of transcription from the latent viral genome. This presents the necessity to identify a viral/host transcriptional activation interface other than the well-defined VP16/Octl/HCF/taatgarat complex. Although the IE gene ICP0 has been viewed as the likely candidate to "replace" VP16 in initiating the lytic cycle, and studies of ICP0 function have revealed much about the role of this protein in modifying the host cell environment, there remains no evidence that ICP0 initiates entry into the lytic cycle. We have generated compelling data, both phenotypic and biochemical that marks VP16 as a critical player in the initiation of reactivation. This key observation calls for a focused examination of the mechanism of upregulation of viral IE gene transcription from latency. The following specific aims will guide us through a systematic dissection of (1) the role of VP16 during the initiation of reactivation; (2) the VP16 promoter elements required for its stress driven upregulation; and (3) the role of viral genome structure in the regulation of transcriptional silencing and initiation of reactivation. The development and refinement of methodologies to facilitate routine examination and quantification of the events occurring in individual neurons in the ganglia have been fundamental to our progress. These analytical tools together with precision engineering of the viral genome and advances in the analysis of chromatin structure will be utilized for these studies. [unreadable] [unreadable]